New Pyranone Derivatives and Sesquiterpenoid Isolated from the Endophytic Fungus Xylaria sp. Z184

The fungus Xylaria sp. Z184, harvested from the leaves of Fallopia convolvulus (L.) Á. Löve, has been isolated for the first time. Chemical investigation on the methanol extract of the culture broth of the titles strain led to the discovery of three new pyranone derivatives, called fallopiaxylaresters A–C (1–3), and a new bisabolane-type sesquiterpenoid, named fallopiaxylarol A (4), along with the first complete set of spectroscopic data for the previously reported pestalotiopyrone M (5). Known pyranone derivatives (6–11), sesquiterpenoids (12–14), isocoumarin derivatives (15–17), and an aromatic allenic ether (18) were also co-isolated in this study. All new structures were elucidated by the interpretation of HRESIMS, 1D, 2D NMR spectroscopy, and quantum chemical computation approach. The in vitro antimicrobial, anti-inflammatory, and α-glucosidase-inhibitory activities of the selected compounds and the crude extract were evaluated. The extract was shown to inhibit nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells, with an inhibition rate of 77.28 ± 0.82% at a concentration of 50 μg/mL. The compounds 5, 7, and 8 displayed weak antibacterial activity against Staphylococcus areus subsp. aureus at a concentration of 100 μM.


Introduction
Natural products (NPs) have always been an indispensable source of new drugs [1].As an important source of NPs with novel structures and high-value biological activities, plant endophytic fungi have always been attracting broad attention from natural product chemists and pharmacologists [2,3].Xylariaceae is one of the largest, most commonly encountered, and highly diverse fungal families of the Ascomycota [4].The genus Xylaria, belonging to the family Xylariaceae, is medicinal fungi commonly found in decaying plant tissues and is widely distributed in temperate, tropical, and subtropical regions [5,6].So far, more than 200 bioactive compounds (>100 new ones) were isolated from Xylaria, including cytochalasins, α-pyrones, cyclopeptides, terpenoids, lactones, and succinic acid derivatives [7].Furthermore, the secondary metabolites produced by species of Xylaria have been found to exert a wide range of biological activities, such as anti-inflammatory, antifungal, antibacterial, anti-tumor, and α-glucosidase-inhibitory activities [7,8].

Results and Discussion
Compound 1, named fallopiaxylarester A, was obtained as a white solid.Its molecular formula C12H16O6 was determined by the HRESIMS molecular ion peak at m/z 279.0837 [M + Na] + (calcd for C12H16O6Na, 279.0839).The IR absorption bands showed the presence of hydroxyl (3426 cm −1 ) and carbonyl (1733 cm −1 ) functionalities.Detailed comparison of 1 H and 13 C NMR spectra of 1 and 10 revealed that the structure of 1 is almost identical with that of 10, which was supported by the further analysis of 2D NMR spectra (Table 1).The HMBC correlations of 1 from H-2 (δH 5.56) to C-1/C-3/C-4, from H3-12 (δH 3.87) to C-3, from H-4 (δH 6.22) to C-3/C-5/C-6, and from H-6 (δH 4.35) to C-5 and C-8, showed the same settlement as with compound 10 (Figure 2).The main difference between 1 and 10 was found at C-10 of the side chain of the pyranone core, replaced by the fragment of methyl valerate group.This deduction was further identified by the HMBC correlations from H3-11 (δH 3.66) and H2-9 (δH 2.38) to C-10.Thus, the planar structure of 1 was established.Since there was only one chiral center in the molecule, the relative configuration was arbitrarily assigned as 6R*.The absolute configuration of C-6 was subsequently assigned to be R by comparing the optical rotational value  [12].Furthermore, the deduction was also confirmed by a time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) approach.As shown in Figure 3, the Boltzmann-averaged ECD spectrum of (6R)-1 displayed a similar curve compared to the experimental one.Thus, the absolute configuration at C-6 in 1 was unambiguously assigned as 6R (Figure 1).
The secondary metabolites generated by stains of Xylaria usually show obvious antiinflammatory and antifungal activities [7,8].In this case, compounds 2-10 and 15-18 and the crude extract were selected to evaluate the antimicrobial, anti-inflammatory and α-glucosidase-inhibition activities due to the limitation of samples.In antimicrobial assay, compounds 5, 7, and 8 displayed weak activity against Staphylococcus areus subsp.aureus with inhibition ratios of 25.9%, 31.5%, and 25.3% at a concentration of 100 µM.Unfortunately, in anti-inflammatory and α-glucosidase assay, only the crude extract potently inhibited LPS-induced NO production in RAW264.7 mouse macrophages, with an inhibition rate of 77.28 ± 0.82% at a concentration of 50 µg/mL.Although it was cytotoxic at this concentration, reducing the concentration to 6.25 µg/mL abrogated the cytotoxicity (Table 5).

Fungal Material
The fungus Xylaria sp.Z184 was isolated from the leaves of Fallopia convolvulus (L.) Á. Löve collected in Zhuyang Town, Henan province, P. R.China (N 34 • 14 ′ 12 ′′ W 110 • 47 ′ 09 ′′ ) in June 2022.Leaves of F. convolvulus (L.) Á. Löve were processed within 24 h and rinsed with sterile water.On a sterile workbench, after 30 min of ultraviolet light exposure, the leaves underwent sequential treatment with a 5% sodium hypochlorite solution, sterile water, and 75% ethanol, either soaked or rinsed, followed by drying with sterile filter paper.Leaves were trimmed into small squares with sterile scissors and placed into previously prepared PDA monoclonal agar plates, inoculating three petri plates in parallel.These plates were incubated at 30 • C for 3-7 days, until mycelial growth was observed extending from the inside of the tissue block to its surroundings.Distinct morphological colonies were subsequently transferred to new media for continued cultivation.This procedure was repeated until the fungal strains showed uniform growth, leading to the isolation of purified strains (Figure 6).
leading to the isolation of purified strains (Figure 6).
To identify the strains, the standardized operating procedure was performed, which included genomic DNA extraction, 16S/18S amplification, PCR product detection and purification, and comparison of sequencing results with the NCBI-BLAST database (https://www.ncbi.nlm.nih.gov/)accessed on 10 December 2022, using ITS1 and ITS4 primers for both amplification and sequencing.The sequence data for this strain was submitted to the GenBank under accession No. KU645984.The fungal strain was deposited on 20% aqueous glycerol stock in a −80 °C freezer at the School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China.

Fermentation, Extraction, and Isolation
Xylaria sp.Z184 was cultured on potato dextrose agar for 5 days at 28 °C to prepare the seed culture.The cultured agar plates were cut into small pieces, which were then inoculated into 30 previously autoclaved Erlenmeyer flasks (350 mL), each containing 50 g of rice and 45 mL of distilled water.All flasks were incubated at 28 °C for 40 days.Cultural media was extracted with methanol four times, and the solvent was evaporated under reduced pressure at 45 °C.Then the extract was suspended in water and extracted four times with ethyl acetate.The combined ethyl acetate layers were concentrated under reduced pressure to yield a brown extract (7.5 g).
The crude extract was chromatographed on Sephadex LH-20 (MeOH) to give eight fractions (Fr.1-Fr.8).Fr.To identify the strains, the standardized operating procedure was performed, which included genomic DNA extraction, 16S/18S amplification, PCR product detection and purification, and comparison of sequencing results with the NCBI-BLAST database (https: //www.ncbi.nlm.nih.gov/)accessed on 10 December 2022, using ITS1 and ITS4 primers for both amplification and sequencing.The sequence data for this strain was submitted to the GenBank under accession No. KU645984.The fungal strain was deposited on 20% aqueous glycerol stock in a −80 • C freezer at the School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China.

Fermentation, Extraction, and Isolation
Xylaria sp.Z184 was cultured on potato dextrose agar for 5 days at 28 • C to prepare the seed culture.The cultured agar plates were cut into small pieces, which were then inoculated into 30 previously autoclaved Erlenmeyer flasks (350 mL), each containing 50 g of rice and 45 mL of distilled water.All flasks were incubated at 28 • C for 40 days.Cultural media was extracted with methanol four times, and the solvent was evaporated under reduced pressure at 45 • C. Then the extract was suspended in water and extracted four times with ethyl acetate.The combined ethyl acetate layers were concentrated under reduced pressure to yield a brown extract (7.5 g).

Computational Details (TDDFT-ECD) of 1
The conformational search of (6R)-1 was performed by using torsional sampling (MCMM) conformational searches with an OPLS_2005 force field within an energy window of 21 kJ/mol.Conformers above 1% Boltzmann populations were re-optimized at the B3LYP/6-31G(d) level with the IEFPCM solvent model for methanol.The following TDDFT calculations of the re-optimized geometries were all performed at the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for methanol.Frequency analysis was performed as well to confirm that the re-optimized geometries were at the energy minima.Finally, the SpecDis 1.62 [27] software was used to obtain the Boltzmann-averaged ECD spectra of 1 and visualize the result.
3.6.Biological Assays 3.6.1.Antimicrobial Activity Compounds 2-10 and 15-18, and the crude extract were evaluated for antimicrobial activities against Staphylococcus aureus subsp.aureus and fluconazole-resistant Candida albicans.The antimicrobial assay was conducted according to a previously described method [28].Samples were added into a 96-well culture plate with a maximum test compound concentration of 100 µM.Bacterial liquid was added to each well until the final concentration was 5 × 10 5 CFU/mL.The plate was then incubated at 37 • C for 24 h, and the OD values at 595 nm were measured using a microplate reader.Blank bacterial medium served as control.

Figure 5 .
Figure 5. Energy-minimized structures of 2 and 4 with the key ROESY correlations.
H , Mult (J Hz) a δ C , Type a δ H , Mult (J Hz) b δ C , Type b δ H , Mult (J Hz) c δ C , Type c a

Table 5 .
Inhibitory activities of compounds selected and crude extract on LPS-stimulated NO production.
a All compounds examined in a set of triplicated experiment.b Positive control.